HOXA‐AS2 Epigenetically Inhibits HBV Transcription by Recruiting the MTA1‐HDAC1/2 Deacetylase Complex to cccDNA Minichromosome

Abstract Persistent transcription of HBV covalently closed circular DNA (cccDNA) is critical for chronic HBV infection. Silencing cccDNA transcription through epigenetic mechanisms offers an effective strategy to control HBV. Long non‐coding RNAs (lncRNAs), as important epigenetic regulators, have an unclear role in cccDNA transcription regulation. In this study, lncRNA sequencing (lncRNA seq) is conducted on five pairs of HBV‐positive and HBV‐negative liver tissue. Through analysis, HOXA‐AS2 (HOXA cluster antisense RNA 2) is identified as a significantly upregulated lncRNA in HBV‐infected livers. Further experiments demonstrate that HBV DNA polymerase (DNA pol) induces HOXA‐AS2 after establishing persistent high‐level HBV replication. Functional studies reveal that HOXA‐AS2 physically binds to cccDNA and significantly inhibits its transcription. Mechanistically, HOXA‐AS2 recruits the MTA1‐HDAC1/2 deacetylase complex to cccDNA minichromosome by physically interacting with metastasis associated 1 (MTA1) subunit, resulting in reduced acetylation of histone H3 at lysine 9 (H3K9ac) and lysine 27 (H3K27ac) associated with cccDNA and subsequently suppressing cccDNA transcription. Altogether, the study reveals a mechanism to self‐limit HBV replication, wherein the upregulation of lncRNA HOXA‐AS2, induced by HBV DNA pol, can epigenetically suppress cccDNA transcription.

HepG2 cells were transfected with plasmid expressing HBV DNA pol for 3 days and incubated with 5 µg mL −1 actinomycin D for the indicated times.The stability of AP-2α was detected by real-time PCR.(I) HepG2-NTCP cells were infected with wild-type HBV particles (HBV WT) or HBV DNA pol-deficient viral particles (HBV-ΔHBV DNA pol) for 9 days and incubated with 5 µg mL −1 actinomycin D for the indicated times.The half-life of AP-2α was detected by real-time PCR.(J) RIP assay for the in vivo interaction between AP-2α and Flag-HBV DNA pol.HepG2 cells were transfected with plasmid expressing Flag-HBV DNA pol.RIP assay was performed using anti-Flag antibody at day 3 post transfection.Western blotting analysis were performed to confirm that Flag-HBV DNA pol was immunoprecipitated in the RIP experiments.(K) HepG2 cells were co-transfected with AP-2α expression plasmid and HOXA-AS2 promoter plasmid, and the level of AP-2α associated with HOXA-AS2 promoter was examined by ChIP assay.Western blotting analysis was performed to confirm that AP-2α was immunoprecipitated in the ChIP experiments.

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Figure S2.HOXA-AS2 reduces HBV-induced oxidative damage.(A) After 24 h of HBV inoculation in HepG2-NTCP cells, the cells were transduced with lentivirus expressing vector or HOXA-AS2 for 5 days.The expression level of HOXA-AS2 was

Figure S3 .
Figure S3.HOXA-AS2 formed an RNA-DNA triplex with cccDNA.(A) Schematic representation of exons and transcripts of HOXA-AS2 and its loci on human chromosome 7p15.2 in UCSC Genome browser (GRCh38.p13).(B) In the ChIRP assay, HBV-infected HepG2-NTCP nuclear pellets lysate was sheared by ultrasound.The product of ultrasonication was analyzed by gel electrophoresis and the result showed nuclear lysate was sheared to 100-500 bp fragments.(C) Reactions containing biotin-labeled HOXA-AS2 WT (1/139) or Mut (deletion of the TFO region (45/68) in HOXA-AS2 WT) and a 40-molar excess of HBV DNA were treated with 0.5 U RNase H or 0.5 ng RNase A for 30 min at room temperature.Upon binding to the streptavidin beads, the associated HBV DNA was analysed by real-time PCR.Normalized data were shown as relative fold enrichment to the control group.(D) HBV-infected HepG2-NTCP cells were transfected with the HOXA-AS2 plasmid or its TFO mutant for 5 days.HOXA-AS2 was visualized by RNA-FISH (red), and immunofluorescence staining of HBc (green) in HBV-infected HepG2-NTCP cells was performed.Nuclei were stained with DAPI (blue).Scale bar, 50 µm.FISH stands for fluorescence in situ hybridization.

Figure S4 .
Figure S4.HOXA-AS2 binding MTA1-HDAC1/2 complex.(A) The specific associations of proteins with biotinylated-HOXA-AS2 were detected by streptavidin RNA-pull down assay and further analyzed by sliver staining and mass spectrometry.The HOXA-AS2 specific binding nucleoproteins were analyzed by Gene Ontology (GO) enrichment analysis.(B) After 24 h of HBV inoculation in HepG2-NTCP cells, the cells were transduced with lentivirus expressing vector or HOXA-AS2, or transfected with control locked nucleic acid (LNA) or HOXA-AS2 LNA for 5 days.The expression levels of MTA1, HDAC1 and HDAC2 were detected using real-time PCR and western blot.(C) GAPDH and H3 were used as a marker for cytoplasmic and nuclear fractions, respectively.(D-E) The interaction between MTA1, HDAC1 and

Figure S5 .
Figure S5.Overexpression efficiency of HOXA-AS2.(A) HepG2-NTCP cells with MTA1 knockout were infected with HBV for 24 h and then transduced with lentivirus expressing vector or HOXA-AS2 for 5 days.The expression level of HOXA-AS2 was detected by real-time PCR.

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Figure S8.HOXA-AS2 inhibits cccDNA transcription in vivo.(A) C57BL/6 mice were injected with prcccDNA and pCMV-Cre.After one week, the mice were injected with a lentivirus-packaged vector or HOXA-AS2 for 21 days.HOXA-AS2 was visualized by RNA-FISH (red), and immunofluorescence staining of HBc (green) in mouse liver was performed.Nuclei were stained with DAPI (blue).Scale bar, 50 µm.(B) The structure prediction and alignment of human MTA1 and mouse Mta1 were performed using AlphaFold2.(C) RNA pull-down and western blot were performed to detect the specific associations of HOXA-AS2 with mouse Mta1.

Figure S9 .
Figure S9.HBV DNA pol induces HOXA-AS2 expression via AP-2α.(A) HepG2 cells were transfected with different HBV protein expression plasmids for 3 days, and the expression levels of HBx, HBc, HBV DNA pol and L-HBs were analysed by western blot.(B) HepG2-NTCP cells were infected with wild-type HBV particles (HBV WT) or HBV DNA pol-deficient viral particles (HBV-ΔHBV DNA pol) for 9 days, and the level of HOXA-AS2 was measured using real-time PCR.(C) HepG2 cells were transfected with plasmid expressing Flag-HBV DNA pol for 3 days, and the level of Flag-HBV DNA pol was detected by western blot.(D) HepG2-NTCP cells were infected with wild-type HBV particles (HBV WT) or HBV DNA pol-deficient viral particles (HBV-ΔHBV DNA pol) for 9 days and incubated with 5 µg mL −1 actinomycin D for the indicated times.The half-life of HOXA-AS2 was detected by real-time PCR.(E) HepG2 cells were transfected with HOXA-AS2 promoter and Flag-HBV DNA pol expression plasmid, and the level of Flag-HBV DNA pol was detected by western blot.(F) HepG2 cells were transfected with HOXA-AS2 promoter and Flag-HBV DNA pol expression plasmid, and the enrichment of HBV DNA pol on the HOXA-AS2 promoter was determined by ChIP assay.Western blotting analysis were performed to confirm that Flag-HBV DNA pol was immunoprecipitated in the ChIP experiments.(G) HepG2-NTCP cells were infected with wild-type HBV particles (HBV WT) or HBV DNA pol-deficient viral particles (HBV-ΔHBV DNA pol) for 9 days, and the level of the transcription factor AP-2α was detected by real-time PCR and western blot.(H) HepG2 cells were transfected with plasmid expressing HBV DNA pol for 3 days and